Eukaryotic initiation factor 5A hypusine as a negative regulator of adenosine 2B receptor (A2bAR) through interaction with stem loop sequences within the A2bAR 3'-untranslated region.
Mol Biol Rep
; 50(4): 3099-3109, 2023 Apr.
Article
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| MEDLINE
| ID: mdl-36689050
ABSTRACT
PURPOSE:
The aim of this study was to investigate whether eIF5A hypusine (eIF5AHyp) reduces adenosine 2b receptor (A2bAR) gene expression through interaction with highly structured stem-loop sequences within the A2bAR 3'UTR. METHODS ANDRESULTS:
Based on real-time PCR and western blotting, expression of A2bAR mRNA was significantly decreased upon treatment with eIF5AHyp in mouse embryonic fibroblasts of eIF5A (eIF5A-MEF) and 3T3-L1 cells. Target Scan software and RNAfold web server predicted two different structures formed by stem-loop sequences with overlapping microRNA 27 seed sequences and mutations. The EMSA results showed significantly impaired formation of the wild type (WT) biotin-labeled A2bAR probe (27 base) containing stem loop sequences-eIF5AHyp complex by mutation of stem-loop sequences or by eIF5A non-hypusine (eIF5ALys). The luciferase reporter assay showed that GC7-induced eIF5ALys accumulation increased the activity of pMIR-A2bAR WT containing the same stem-loop sequence in 3T3-L1 cells, whereas the activity with pMIR-A2bAR Mut was increased compared to WT control without dependence on GC7. Oil Red O staining showed that suppression of A2bAR expression (A2bAR siRNA and eIF5AHyp) increased the amount of lipid droplet formation and the mRNA levels of lipid droplet-related genes (C/EBP-ß, PPAR-γ, FABP4, SREBP-1, and Perilipin). In contrast, overexpression of A2bAR (A2bAR vector, eIF5ALys vector, and GC7) significantly decreased the expression of lipid droplet-associated genes and lipid droplet formation.CONCLUSIONS:
eIF5AHyp acts as a negative regulator of A2bAR gene expression through stem loop sequences in A2bAR 3'UTR, allowing differentiation of adipocytes.Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
MicroARNs
/
Fibroblastos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Mol Biol Rep
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos